Tobacco streak virus (TSV) is the type species of the
Ilarvirus genus of the family Bromoviridae (5), and is transmitted
mechanically, through thrips feeding, by pollen (6), and by seed (4). The virus
has a tripartite genome of single-stranded messenger-sense RNA, which encodes
four non-structural proteins and a single structural capsid protein (5). The
movement protein and capsid protein encoding genes are located on the 5' half
and 3' half, respectively, of RNA 3 (5).

Double-stranded ribonucleic acid (dsRNA) was purified from
symptomatic leaf tissue and used as a template for cDNA synthesis as previously
described (2,7). Immunocapture reverse transcription (IC-RT) using sheep
anti-rabbit conjugated magnetic beads incubated with polyclonal rabbit anti-TSV
IgG was also performed as previously described (1,3). TSV specific primers were
used to amplify the movement protein (MP) and capsid protein (CP) genes from
cDNAs synthesized from dsRNA template and immunocaptured virions (1). Both sets
of primers amplified distinct products (Fig. 2). The amplicons were excised,
purified from the agarose, and cloned into pGEM-T vector as previously described
(2, 3). Colonies were screened for inserts which were subsequently sequenced.
Sequences were assembled and subjected to pairwise and multiple sequence
alignments (1,2). Finally, open reading frames (ORFs) were translated using
Genedoc (Genedoc v. 2.6.001).

Ten MP and nine CP clones were sequenced (The Ohio State University
Plant Microbe Genomics Facility) and the ORF sequences deposited in GenBank
(accession numbers KC776130- KC776148). The TSV-Astilbe MP ORF is 870
nucleotides (nt) long and encodes a predicted 289 amino acid (aa) protein. The
clones had 99.4-100% nt sequence identity to one another with a 99.8%
mean, and a 99.0-100% predicted aa identity to one another with a 99.7% mean.
The TSV-Astilbe CP ORF is 714 nt long and encodes a predicted 237 aa protein.
When aligned, two distinct populations of CP clone nt sequences were identified.
Six of nine clones were 99.4-100% identical to one another with a 99.7% mean,
and three of nine clones were 99.7-99.9% identical to one another with a 99.8%
mean. However, the two populations were only 91.9-92.4% identical to each other
with a 92.1% mean. The translated CP ORFs could also be divided into two
distinct populations of predicted aa sequences. Six of nine clones were
98.7-100% identical with a 99.2% mean, and three of nine clones were 99.6-100%
identical with a 99.7% mean. The two populations were only 92.8-93.2% identical
to each another with a 93.1% mean. The two CP populations could also be
distinguished based on the source of the cDNA used for amplification. Five of
five clones from cDNAs from immunocaptured virions were all of the same
genotype, but clones from cDNAs from dsRNA template were of two distinct
genotypes. One of the four clones belonged to the same population as those from
immunocaptured virions. The other three were distinctly different. These results
demonstrate that at least two TSV RNA 3 variants are associated with virus-like
symptoms in Astilbe × arendsii. The MP nt and predicted aa sequences are highly
conserved among all of the sequences analyzed here, but there are clearly two CP
nt and aa sequence variants. Further, when compared to a TSV isolate we recently
reported from Hosta (1) the TSV-Astilbe CP ORF is three nt shorter and the
predicted protein is two aa shorter. The two TSV-Astilbe CP variants had only
88.7% and 90.0% nt sequence identity, and 90.3% and 92.4% aa identity to
TSV-Hosta. BLASTn searches of the NCBI database using default settings with the
TSV-Astilbe MP ORF sequences (100% query coverage) resulted in an 82% match with
accession numbers X00435.1, JX073658.1, and FJ403377.1. BLASTn searches using
default settings with the two TSV-Astilbe CP ORF variants resulted in reciprocal
matches: 98% (accession number X00435.1) and 92% (accession number AM933669.1)
with one variant and 98% (accession number AM933669.1) and 92% (accession number
X00435.1) with the second variant.

To our knowledge, the results presented here represent the first
confirmed report of TSV infecting Astilbe × arendsii. They also
demonstrate that there are two CP nt sequence variants that translate into two
CP aa sequence variants. It is interesting to note that only one of the
variants was detected by IC-RT-PCR but both were detected from cDNAs from dsRNA
template. That we only detected the second CP ORF variant using a strictly
nucleic acid based detection method may be significant and might suggest that
the antibodies used in this study aren’t detecting the second variant, but an
analysis of a larger sampling of clone sequences would be necessary to rule out
that possibility. Commercial perennial growers should benefit from this
work by gaining awareness of TSV as an emerging threat to ornamentals in
general, and a new threat to Astilbe production in particular.